Flow cytometry is a laser-based, biophysical technology where fluorescent molecules coupled to cells are passed through a flow cell and excited by a set of lasers. The fluorescence is collected and separated into different channels with specific detection wavelength, converted to electrical signals, and analyzed using a computer. Multi-color flow cytometry, such as three color flow cytometry uses fluorophores with different excitation and emission wavelengths for identification of different staining of the biological samples. More specifically, the excitation light is delivered to the flow cell by beam-shaping, steering, and guiding optical components. Conventionally, such components are arranged so that light from all lasers pass through a same set of beam shaping, steering and guiding components before reaching the flow cell. Such beam shaping optics can include, for example, achromatic lenses (cylindrical or spherical) to accommodate different wavelengths of different lasers. In addition, the beam-steering/guiding components direct all light beams to the center of the flow cell, either at a same position or at different positions along the flowing direction of the sample. However, a limitation of this approach is that by providing a same set of optics for the set of lasers, the beam shaping and beam guiding components are compromised in that they shape and steer light beams of different wavelengths for somewhat sub-optimum beam sizes to sub-optimum focus-locations. Thus, there is a need for an approach to optical illumination that improves beam size and focusing point. In addition, the beam size and focusing point should be able to be adjusted independently for each laser.
In addition to fluorescence, two other types of light scatter are measured in flow cytometery, namely side scatter and forward scatter. Forward scatter (FSC) is considered a low angle scatter and is roughly proportional to the diameter of the cell. Therefore FSC is useful in identifying certain cell subpopulations from others based on cell size. Since forward scatter is typically measured along a same path as beam propagation, an optical beam obscuration bar is conventionally added behind the flow cell but along the optical path of laser beam propagation to block the unscattered, high-intensity raw laser beam. A challenge in obscuration bar design is that it should block the unscattered raw laser beam but permit passage of low angle forward scattered light for detection. Conventionally, the obscuration bar is either rectangular or cross-shaped. Such geometry is generally designed empirically, with some geometries blocking too much forward scatter light yet other designs lacking sufficient blocking of unscattered light. Therefore, there is also a need for an improved optical beam obscuration bar that improves the balance of blocking unscattered light while permitting passage of forward scatter light.
In another flow cytometer, fluorescent light is collected from the particles (e.g. cells) flowing through the center of the flow cell. Complex design including multiple-lenses positioned at accurate locations relative to each other and relative to flow cell is employed to collect fluorescent light from the particles. Such collection optics is expensive to make, difficult to align and adjust. For many situations, the light collection efficiency is limited. Therefore, there is also a need for an improved collection optics that is simple in design, comprising fewer optic components and has high light-collection efficiency.